Abstract

Conventional polymerase chain reaction (PCR) performed in a continuous aqueous phase often shows an inability to detect ultra-low DNA concentration. Droplet digital PCR, where DNA polymerization happens in isolated water droplets of water-in-oil (W/O) emulsions, could significantly improve the detection limit. However, water droplet breakups under multiple thermal cycles necessary for DNA polymerization is preventing the wide-spread application of such technology. The present research aims to develop thermally stable coarse W/O emulsions that could act as DNA microreactors. Span 80, polyglycerol polyricinoleate (PGPR), or sodium bis(2-ethylhexyl) sulfosuccinate (AOT) were dissolved in mixtures of light and heavy mineral oils at different concentrations. The aqueous phase was selected based on PCR protocol, without the presence of enzymes and DNA. Emulsions prepared without bovine serum albumin (BSA) in the aqueous phase was used as a control to understand its effect on stability. Emulsions were formed using a vortex mixer for 2 minutes at 3200 rpm. Only the emulsions without visible aqueous phase separation were exposed to PCR thermal cycling. The volume mean diameters of water droplets were calculated by image analysis. Span 80-stabilized emulsions destabilized upon thermal cycling, while PGPR-stabilized emulsions remained stable with an increase in water droplet size. AOT was able to form thermally stable emulsions, with an average droplet size around 119 µm without any significant change. The influence of emulsifiers concentration, surface saturation, molecular interaction with BSA at the interface, and continuous oil phase viscosity was considered to explain the mechanism of emulsion formation and thermal stability.

摘要

在连续水相中进行的常规聚合酶链反应 (PCR) 通常无法检测到超低浓度的 DNA。液滴数字 PCR，其中 DNA 聚合发生在油包水 (W/O) 乳液的孤立水滴中，可以显着提高检测限。然而，DNA 聚合所需的多次热循环下的水滴破裂阻碍了这种技术的广泛应用。本研究旨在开发可用作 DNA 微反应器的热稳定粗 W/O 乳液。将 Span 80、聚甘油聚蓖麻油酸酯 (PGPR) 或双(2-乙基己基) 磺基琥珀酸钠 (AOT) 溶解在不同浓度的轻质和重质矿物油的混合物中。水相是根据 PCR 方案选择的，不存在酶和 DNA。在水相中不含牛血清白蛋白 (BSA) 的乳液用作对照，以了解其对稳定性的影响。使用涡旋混合器以 3200 rpm 搅拌 2 分钟形成乳液。只有没有可见水相分离的乳液暴露于 PCR 热循环。通过图像分析计算水滴的体积平均直径。Span 80 稳定的乳液在热循环时不稳定，而 PGPR 稳定的乳液随着水滴尺寸的增加而保持稳定。AOT 能够形成热稳定的乳液，平均液滴尺寸约为 119 µm，没有任何显着变化。乳化剂浓度、表面饱和度、界面处与 BSA 的分子相互作用的影响，